DESCRIPTION: The Lundblad lab is using a genetic approach in the budding yeast Saccharomyces cerevisiae to identify potential components or positive regulators of telomerase. During the previous grant period the lab conducted a genetic screen for yeast mutants that displayed a similar phenotype to the previously identified est1- mutant. This mutant displays progressive telomere shortening and a senescence phenotype. The genetic screen resulted in identification of two new EST genes (EST2 and EST3) as well as a novel est-like mutation in a gene previously implicated at the telomere (CDC13). The CDC13 and EST gene products are essential in vivo for telomere replication but are dispensable in vitro for telomerase activity. Thus, they may be essential regulators of telomerase rather than part of the core enzyme. Since Est1p and Cdc13p are both single-strand telomere binding proteins, the PI proposes that these two proteins function to mediate access of telomerase to the chromosomal terminus. Est2p and Est3p could act similarly as components of telomeric chromatin, or as other in vivo regulators of telomerase. This application proposes to take two inter-related approaches to further analysis of the EST/TLC1 pathway for telomere replication. The first is to extensively characterize each EST gene with the long term goal of determining what each individual protein is doing at the telomere and using this information to ultimately reconstitute telomerase activity in vivo. Specifically, the lab will analyze the biochemical properties of the Est1 protein; look for potential interactions between the individual Est proteins, as well as between the Est proteins and other gene products; determine whether the Est proteins form a complex and are components of either telomeric chromatin or telomerase; reconstitute yeast telomerase activity. In a parallel approach, the lab will conduct several extensive mutant screens designed to identify additional genes required for telomere function in yeast including additional EST genes. In this aim the lab will perform a new mutant screen designed to identify additional genes in the EST/TLC pathway for telomere replication; screen the 4900 viable yeast null mutations for changes in telomere length; characterize and clone the genes mutated in a new collection of 25 short and long telomere mutants. The genes identified in these screens will be incorporated into the ongoing genetic and biochemical experiments with the existing EST genes.